Method and associated device for rapid detection of target biomolecules with enhanced sensitivity

ABSTRACT

A rapid detection method of a target biomolecule comprising an antigenic moiety is provided. The method includes providing a source biological sample comprising the target biomolecule; contacting the source biological sample to an ion-exchange medium; eluting the captured-target biomolecule from the ion-exchange medium as an eluate, and loading the eluate to a rapid diagnostic testing device comprising an antibody. The eluate comprises a concentrated form of the biomolecule in a solution having a salt concentration greater than 150 mM. A concentration of the target biomolecule in the eluate is in a range from about 2× to 25× compared to a concentration of the biomolecule in the source biological sample. The target biomolecule binds to the antibody under the salt concentration of greater than 150 mM. A device for rapid detection of target biomolecule is also provided.

This application relates generally to a rapid detection method ofinfectious disease. In a particular aspect, the application relates to arapid detection method and an associated device for rapid diagnostictesting of a target biomolecule.

BACKGROUND

A rapid detection method of analytes or target biomolecules performed byemploying a rapid diagnostic test (RDT) is relatively less timeconsuming and less labor intensive as compared to conventional methods.Rapid diagnostic tests (RDTs) have been used for detection of variousinfectious diseases. The RDTs are suitable for preliminary and/oremergency medical screening, for example, for use in medical facilitieswith limited resources, and offer a useful alternative to microscopy insituations where reliable microscopic diagnosis facility is notavailable or is not immediately available. RDTs also allow point of care(POC) testing in primary care. RDTs can be performed independent oflaboratory equipment by minimally trained personnel, and are adapted todeliver instant results. RDTs provide results within 2 hours to 10minutes. An RDT employs a dipstick or cassette format for testing abiological specimen, such as a urine sample. For testing, the biologicalspecimen collected from a patient is applied to a sample pad on a teststrip (or card) of the RDT dipstick or cassette along with certainreagents. Depending on the type of test that is being conducted, after adetermined period of time, presence or absence of specific bands in atest strip window indicates whether a certain antigen of interest ispresent in the biological specimen, such as a patient's sample.Generally, a drop of the biological specimen is added to the RDT devicethrough a sample well, and then a buffer is usually added through abuffer well. The buffer carries the biological specimen along the lengthof the RDT device.

Among major infectious diseases, tuberculosis is different in that itlacks accurate rapid point-of-care diagnostic tests. Inefficientdetection methods and lack of timely treatment of tuberculosis are majorcauses of failure to control the spread of tuberculosis. Laboratorybased diagnostic tests for detecting tuberculosis are currentlyavailable, however these tests need multiple investigations over aperiod of weeks or months. Multiple new diagnostic tests have recentlybeen developed for detecting active tuberculosis, latent tuberculosisinfection, and identifying drug-resistant strains of Mycobacterialtuberculosis. However, a robust point-of-care test with high accuracy,greater accessibility, reduced cost and complexity is desirable forearly detection of tuberculosis. Further, an effective method fordiagnosing extra-pulmonary mycobacterial tuberculosis infections, whichare on the rise in HIV-positive subjects, is also highly desirable.

Prior methods for detecting surface polysaccharides (LAM) usingdifferent body fluids, such as serum, urine or sputum, have beeninvestigated, but have proven ineffective. For example, prior studieswith urine sample required extensive sample processing and manipulation,rendering such methodologies complex and cumbersome, specifically in thefield.

BRIEF DESCRIPTION

In some embodiments, a rapid detection method of a biomolecule isprovided. The method comprises providing a source biological samplecomprising the biomolecule; contacting the source biological sample toan ion-exchange medium comprising one or more ligands to capture thebiomolecule and form a captured-biomolecule; eluting thecaptured-biomolecule from the ion-exchange medium as an eluate, andloading the eluate to a rapid diagnostic testing device comprising anantibody. The biomolecule comprises an antigenic moiety. The eluatecomprises a concentrated form of the biomolecule in a solution. Aconcentration of the biomolecule in the eluate is in a range from about2× to 25× compared to a concentration of the biomolecule in the sourcebiological sample. The solution has a salt concentration greater than150 mM. The biomolecule binds to the antibody under the saltconcentration of greater than 150 mM.

In some other embodiments, a method for rapid diagnostic testing of asource urine sample comprising tuberculosis-lipoarabinomannan (TB-LAM)is provided. The method comprises (a) concentrating the TB-LAM by:diluting the source urine sample by at least 2× compared to the sourceurine sample to form a diluted urine sample; contacting the dilutedurine sample to an anion-exchange medium to capture the TB-LAM of thediluted urine sample; capturing the TB-LAM of the diluted urine sampleby the anion-exchange medium; and eluting the captured-TB-LAM from theanion-exchange medium as a concentrated form of TB-LAM in an eluateunder a salt concentration of at least 1M. A concentration of the TB-LAMin the eluate is in a range from about 2× to 25× compared to aconcentration of the TB-LAM in the diluted urine sample. The method alsocomprises (b) loading the eluate comprising the concentrated form of theTB-LAM of step (a) to a lateral flow assay device comprising aTB-LAM-specific antibody for binding the concentrated form of theTB-LAM. The eluate is loaded without any dilution, and wherein theTB-LAM binds to the TB-LAM-specific antibody under the saltconcentration of at least 1M.

Another embodiment of a device is also provided. The device comprises a)a concentrator unit comprising an anion exchange medium to concentratean antigen from a biological sample and generate a concentrated form ofthe antigen; and b) an immune-based assay unit comprising an antibody tobind the concentrated form of the antigen received from the concentratorunit, wherein the concentrator unit is operatively coupled to theimmune-based assay unit to allow loading of the concentrated form of theantigen from the concentrator unit to the immune-based assay unit.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a flow chart illustrating one embodiment of a rapid detectionmethod of a biomolecule.

FIG. 2A is a schematic drawing of a perspective view of one embodimentof a device for rapid detection of a biomolecule.

FIG. 2B is a schematic drawing of a perspective view of anotherembodiment of a device for rapid detection of a biomolecule.

FIG. 3 is an image illustrating different commercially available rapiddiagnostic test strips showing presence of TB-LAM in a source biologicalsample.

FIG. 4 is an image depicting different commercially available rapiddiagnostic test strips showing presence of TB-LAM in a flow through.

FIG. 5 is an image illustrating different commercially available rapiddiagnostic test strips showing presence of TB-LAM in an eluate.

FIG. 6 is an image illustrating different rapid diagnostic test stripsshowing concentrated (15×) form of TB-LAM in different eluate samplescompared to a control with no TB-LAM.

FIG. 7 is an image illustrating different LFA test strips showingconcentrated (25×) form of TB-LAM in different eluate samples elutedfrom the ion-exchange medium as compared to control samples which arenot contacted with the ion-exchange medium.

DETAILED DESCRIPTION

A rapid detection method and associated devices are provided, whichovercome difficulties in the currently known methods or devices byproviding enriched target biomolecules (e.g., mycobacterial antigens) ina biological sample collected from a subject. The biological sampleenriched with target biomolecule further enhances sensitivity of therapid diagnostic tests. In accordance with certain embodiments, a rapiddetection method of target biomolecules present in a biological sampleemploys rapid diagnostic tests (RDTs) or rapid diagnostic test (RDT)devices. Rapid diagnostic testing devices produce a visible band on arapid diagnostic testing device by capturing a target biomolecule(antigens) using antibodies, such as dye-labeled antibodies. For rapiddiagnostic testing, in one aspect, the dye-labeled antibody or conjugateparticle-coupled antibody binds to the target biomolecule (antigen),such as a tuberculosis biomarker. The resultant targetbiomolecule-antibody complex may further be captured by a secondaryantibody or a conjugate particle-coupled secondary antibody forming avisible band (test line) in a result window of the rapid diagnostictesting device.

To more clearly and concisely describe the subject matter of thedisclosed application, the following definitions are provided forspecific terms, which are used in the following description and theappended embodiments. Throughout the specification, exemplification ofspecific terms should be considered as non-limiting examples.

The singular forms “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise.

Approximating language, as used herein throughout the specification andclaims, may be applied to modify any quantitative representation thatcould permissibly vary without resulting in a change in the basicfunction to which it is related. Accordingly, a value modified by a termsuch as “about” is not to be limited to the precise value specified. Insome instances, the approximating language may correspond to theprecision of an instrument for measuring the value. Where necessary,ranges have been supplied, and those ranges are inclusive of allsub-ranges there between.

As used herein, the term “rapid diagnostic test” or RDT refers to adevice or a test of a biological sample, which can be carried out at thepoint of care to obtain fast diagnosis. Rapid diagnostic testing devicesor rapid diagnostic testing devices employ rapid diagnostic tests thatare medical diagnostic tests that are quick and easy to perform and canbe carried out even in the absence of laboratory techniques such asmicroscopy, enzyme-linked immunosorbent assay (ELISA) or polymerasechain reaction (PCR). By way of a non-limiting example, rapid diagnostictesting for tuberculosis typically require about 30 minutes from thetime of sample collection to the time of obtaining a result. It shouldbe noted that time required for a rapid diagnostic testing depends onvariables, such as the type of sample, the amount of sample, the natureof the analyte, and the like.

Embodiments of a rapid detection method of a target biomolecule using arapid diagnostic testing device are presented herein. The rapiddetection method and the rapid diagnostic testing device are compatiblewith equipment-free, point of care analyte-separation and detectionprocess. For example, the rapid diagnostic testing device provides rapiddiagnostic testing by immunochromatographic separation and detection ofbiomolecule from a biological sample, such as urine. Improvedsensitivity of a rapid diagnostic testing is desired, however, an earlydetection of tuberculosis (TB) is made complicated by frequent falsepositive tests and a huge variability of band intensity particularly inthe very low concentration range.

According to embodiments of the present technique, a rapid detectionmethod of a target biomolecule comprises providing a source biologicalsample comprising the target biomolecule, contacting the sourcebiological sample to an ion-exchange medium and form a captured-targetbiomolecule, eluting the captured-target biomolecule from theion-exchange medium as an eluate comprising a concentrated form of thetarget biomolecule in a solution, and loading the eluate to an rapiddiagnostic testing device comprising an antibody. The target biomoleculeof the source biological sample comprises an antigenic moiety. Theion-exchange medium comprises one or more ligands to capture the targetbiomolecule. A concentration of the target biomolecule in the eluate isin a range from about 2× to 25× compared to a concentration of thetarget biomolecule in the source biological sample. The eluate comprisesthe concentrated form of the target biomolecule in the solution, wherethe solution has a salt concentration greater than 150 mM. The targetbiomolecule binds to the antibody of the rapid diagnostic testing deviceunder the salt concentration of greater than 150 mM.

In one or more embodiments, the target biomolecule is a glycolipid. Thetarget biomolecule may comprise a lipoarabinomannan (LAM), which is acomplex lipopolysaccharide antigen composed of mannose and arabinoseresidues forming a highly branched and complex structure. In someembodiments, the target biomolecule is a tuberculosis-lipoarabinomannan(TB-LAM). In such embodiments, if the TB-LAM is detected in thebiological sample (such as urine), it is concluded that the biologicalsample is collected from a TB-positive subject. LAM is a virulencefactor associated with Mycobacterium tuberculosis, the bacteriaresponsible for TB. The bacterium survives in the human reservoir byundermining host resistance and acquired immune responses by inhibitingT-cell proliferation and macrophage microbicidal activity. It isbelieved that as LAM outflows into the circulation in an active TBinfection and passes through the kidneys, LAM can be detected in theurine sample of a TB patient reflecting the level of mycobacterialburden. TB-LAM is typically found in human urine at very lowconcentration, such as in picogram level. The RDTs are prone to givefalse positive results to identify TB-LAM at such low concentrationlevels. To achieve desired results using RDTs, a concentrated form ofTB-LAM may be used.

Referring now to FIG. 1, a method 10 for rapid detection of a targetbiomolecule broadly encompasses two processes: a first process describedin blocks 12-16 that includes concentrating the target biomolecule toincrease sensitivity of the rapid diagnostic test, such asimmuno-chromatographic antigen-detection tests, and a second processdescribed in block 18 that includes loading the concentrated form of thetarget biomolecule to a rapid diagnostic testing device for detection ofthe target biomolecule. At block 12, a source biological samplecomprising the target biomolecule is provided. The source biologicalsample may be collected from a patient suffering from an infectiousdisease, such as tuberculosis (TB). In a non-limiting example, thesource biological sample may be a urine sample collected from a TBpatient for detection of TB-LAM. At block 14, the source biologicalsample is contacted with an ion-exchange medium. Further, at block 16,the captured target biomolecule is eluted from the ion-exchange mediumas an eluate, where the eluate comprises a concentrated form of thetarget biomolecule in a solution. At block 18, the eluate comprising theconcentrated form of the biomolecule in the solution is loaded to arapid diagnostic testing device. The eluted solution referred to hereinas an “eluate,” contains a concentrated form of the target biomolecule(e.g., TB-LAM) in a solution having a salt concentration greater than150 mM. The rapid diagnostic testing device comprises an antibody thatfacilitates detection of the concentrated form of the target biomoleculeby an immuno-chromatographic antigen-detection test.

The target biomolecule present in the source biological sample providedat block 12 may include different biomarkers present in the sourcebiological sample, e.g., biomarkers for infectious disease such as TB.The source biological sample may comprise a plurality of constituentsincluding the target biomolecule having an antigenic moiety, such asTB-LAM.

Optionally, in one or more embodiments, the method 10 further comprisesdiluting the source biological sample, as shown in block 20. In someembodiments, the source biological sample (e.g., urine) comprising thetarget biomolecule (e.g., TB-LAM) is diluted at least by 2× to form adiluted biological sample before contacting the source biological sampleto the ion-exchange medium at block 14. In some other embodiments, thesource biological sample (e.g., urine) comprising the biomolecule isdiluted by 4× to form a diluted biological sample before contacting thesource biological sample to the ion-exchange medium. For example, aurine sample collected from a subject is diluted by 4× with a buffer toreduce salinity of the urine sample. By reducing the salinity of theurine sample, binding efficiency of the target biomolecule of the urinesample to the ion-exchange medium may be enhanced. The source biologicalsample may be a urine sample collected from a TB patient for detectionof TB-LAM using immuno-chromatographic antigen-detection tests.

The sensitivity of the immuno-chromatographic antigen-detection testsrely on concentration of the target biomolecules (or antigens) presentin the particular source biological sample. To achieve a desiredsensitivity of detection, at block 14, a concentrated form of the targetbiomolecule (such as TB-LAM) is generated by contacting the sourcebiological sample to an ion-exchange medium comprising one or moreligands to capture the target biomolecule. The step of contacting thesource biological sample to the ion-exchange medium allows the targetbiomolecule to bind to the one or more ligands of the ion-exchangemedium and form a captured-target biomolecule. The captured-targetbiomolecule refers to the target biomolecule that is bound to a ligandof the ion-exchange medium.

In certain embodiments, the step of contacting the source biologicalsample to the ion-exchange medium includes following sub-steps, such as,but not limited to, applying or loading the source biological sample tothe ion-exchange medium, binding or capturing of the target biomoleculeto the ligand of the ion-exchange medium, and flowing (or discarding)the unbound constituents of the source biological sample through theion-exchange medium. In some embodiments, the sub-steps of binding thetarget biomolecule and flowing the unbound constituents may be performedsimultaneously. As used herein, the phrase “capturing of the targetbiomolecule by the one or more ligands” refers to binding of the targetbiomolecule to the one or more ligands. The unbound constituents of thesource biological sample, optionally, may be collected as a flowthrough.

As noted above, at block 16, the captured-target biomolecule, such asTB-LAM, is retrieved by elution of the captured-target biomolecule fromthe ion-exchange medium as an eluate, the eluate comprises aconcentrated form of the target biomolecule in a solution. One or morebuffers may be used for eluting the captured-target biomolecule form theion-exchange medium. In some embodiments, the captured-targetbiomolecule is eluted by applying a buffer (e.g., an elution buffer)having a salt concentration greater than 150 mM. In some suchembodiments, the eluate containing a concentrated form of the targetbiomolecule, such as TB-LAM, also contains salt that is carried overfrom the elution buffer. By way of example, a concentrated form ofTB-LAM is eluted from the anion exchange medium, such as an anionexchange membrane (e.g., Q⁺ adsorber) by a small volume such as ˜100 uLof buffer having a salt concentration of 1M.

Optionally, in certain embodiments, one or more of a source biologicalsample, a flow through, and an eluate after elution from theion-exchange medium are optionally collected to test to determinepresence or absence of target biomolecule, as shown at blocks 22, 24 and26, respectively. The test to determine presence of the targetbiomolecule in each of the source biological sample, flow through, andeluate is performed by using enzyme linked immunosorbent assay, rapiddiagnostic test, or both. In a non-limiting example, a commerciallyavailable enzyme linked immunosorbent assay (ELISA) kit, a commerciallyavailable rapid diagnostic test kit, or a combination thereof may beused to determine the presence of the target molecule at one or moreblocks 22, 24, and 26. For example, optionally, at block 22, the sourcebiological sample may be tested for the target biomolecule by applyingthe source biological sample to a commercially available rapiddiagnostic test kit. Further, at block 24, the flow through may beoptionally collected for testing for the presence or absence of thetarget biomolecule (e.g., TB-LAM). At block 26, the eluate may also beoptionally tested for presence or absence of the target biomolecule byapplying the eluate to a commercially available rapid diagnostic testkit.

In some embodiments of block 16, the elution of the captured-targetbiomolecule from the ion-exchange medium is effected under a saltconcentration in a range from about 0.5M to about 2M. In some otherembodiments, the elution of the captured-target biomolecule from theion-exchange medium is effected under a salt concentration in a rangefrom about 0.75M to about 1.75M. In certain embodiments, the elution ofthe captured-biomolecule from the ion-exchange medium is effected undera salt concentration in a range from about 1M to about 1.5M. As such,the eluate, which is directly loaded to the rapid diagnostic testingdevice, comprises a concentrated form of the biomolecule (TB-LAM) in abuffer of high salt concentration.

As noted, at block 18, the eluate comprising the concentrated form ofthe target biomolecule in the solution is loaded to a rapid diagnostictesting device, wherein the rapid diagnostic testing (RDT) broadlyincludes enzyme linked immuno-sorbent assay (ELISA), lateral flow assays(LFAs), and/or flow through assays (FTAs). In one embodiment, the rapiddiagnostic testing employed for the method of rapid detection of targetbiomolecule is a lateral flow assay (LFA). Sensitivity of theimmunochromatographic antigen-detection tests further rely on bindingefficiency of an antigen (here, target biomolecule, such as TB-LAM) toan antibody and the stability of the antigen-antibody complex. Toprovide a better binding efficiency of a target biomolecule to anantibody, a concentrated form of the target biomolecule (such as TB-LAM)present in the eluate is directly loaded to the rapid diagnostic testingdevice comprising an antibody to form a stable targetbiomolecule-antibody complex. In some embodiments, the eluate iscollected using a buffer solution having a salt concentration greaterthan 150 mM in a volume of 70-100 μL and directly loaded to the rapiddiagnostic testing device, without any dilution. A small volume ofeluate, such as 70-100 μL, comprising the concentrated form of thetarget biomolecule in a buffer of high salt concentration may be loadedto the rapid diagnostic testing device comprising an antibody. In somecases, 50-100 μL of eluate comprising the concentrated form of thebiomolecule in a buffer of high salt concentration may also be loaded tothe RDT device comprising an antibody. In some other embodiments, theeluate may be diluted before loading the eluate to the rapid diagnostictesting device, however the dilution is maintained such that it does notaffect the binding efficiency of the antigen and antibody, as well asthe stability of the antigen-antibody complex.

With respect to block 18, for rapid detection of a target biomolecule,the efficient binding of the target biomolecule-antibody occurs under asalt concentration greater than 150 mM, which is an unexpected result.In some embodiments, the target biomolecule binds to the antibody undera salt concentration in a range from about 0.5M to about 2M. In someother embodiments, the target biomolecule binds to the antibody under asalt concentration of about 1.3M. The binding of an antigen (targetbiomolecule) to an antibody under such salt concentration (greater than150 mM) is not reasonably expected considering standard protocol ofimmunoprecipitation. Generally, a salt concentration of greater than 150mM is the range where standard antigen-antibody binding assays are notexpected to be successful. As the antibodies are designed to bind to atarget antigen inside a body of a living subject under a physiologicalcondition of salt concentration, such as less than 150 mM salt, the saltconcentration of less than 150 mM is selected for standard protocol ofimmunoprecipitation for successful antigen-antibody binding assay. Theart recognized buffers for complete elution of antibodies from theantigen-antibody complexes include 100 mM Glycine, pH2.5 (acidic pH), 1MTriethanolamine; TEA (basic pH), 4M MgCl₂ (high salt), 1M NaCl/PBS (highsalt). A salt concentration of 1M is currently used to completely detachan antibody from an antigen-antibody complex. Considering these facts,an antigen-antibody interaction on the rapid diagnostic testing deviceunder such salt concentration of greater than 150 mM is quiteunexpected. The Examples 1-3, FIGS. 5-7 also demonstrate the desiredantigen-antibody interaction under a salt concentration in a range fromabout 0.5M to about 2M. In one example, the biomolecule, such as TB-LAM(antigen) binds to the antibody under the salt concentration of 1.3M(Examples 1-3, FIGS. 5-7), which is an unexpected result.

The target biomolecule binds to the antibody on a rapid diagnostictesting device, wherein the rapid diagnostic testing method comprisesgenerating a signal on antigen-antibody binding. For example, on bindingof the TB-LAM to the TB-LAM-specific antibody, such as reporter-linkedTB-LAM specific antibody, a signal is generated in case of exceeding apre-determined threshold value for a concentration of the TB-LAM. Thehigher signal intensity is advantageous for detection of targetbiomolecule because generally RDTs rely on visually detected changes incolor of the test region on an immune-based assay unit. A faint colorchange is not visually detectable and could lead to a false negativeresult on the RDT device. The method 10 ensures generating aconcentrated form of target biomolecule, such as TB-LAM and loading theconcentrated form of the target biomolecule TB-LAM to the RDT devicecomprising TB-LAM-specific antibody to generate better signal intensitycompared to the commercially available RDTs. The method 10 is alsoeffective for diagnosing extra-pulmonary mycobacterial infections for asubject having co-infection of TB and human immune deficiency virus(HIV).

In certain embodiments, the rapid detection method described herein maybe used to detect tuberculosis-lipoarabinomannan (TB-LAM) in a urinesample. The method comprises (a) concentrating the TB-LAM by: dilutingthe source urine sample by at least 2× compared to the source urinesample to form a diluted urine sample; contacting the diluted urinesample to an anion-exchange medium to capture the TB-LAM of the dilutedurine sample; capturing the TB-LAM of the diluted urine sample by theanion-exchange medium; and eluting the captured-TB-LAM from theanion-exchange medium as a concentrated form of TB-LAM in an eluateunder a salt concentration of at least 1M. A concentration of the TB-LAMin the eluate is in a range from about 2× to 25× compared to aconcentration of the TB-LAM in the diluted urine sample. The method alsocomprises (b) loading the eluate comprising the concentrated form of theTB-LAM of step (a) to a lateral flow assay device comprising aTB-LAM-specific antibody for binding the concentrated form of theTB-LAM. The eluate is loaded without any dilution, and wherein theTB-LAM binds to the TB-LAM-specific antibody under the saltconcentration of at least 1M.

In certain embodiments, the device structure is described herein togenerally correlate the method steps to the device components. A devicefor rapid detection of a target biomolecule from a source biologicalsample is provided. The device comprises a) a concentrator unitcomprising an anion exchange medium to concentrate an antigen from abiological sample and generate a concentrated form of the antigen; andb) an immune-based assay unit comprising an antibody to bind theconcentrated form of the antigen received from the concentrator unit,wherein the concentrator unit is operatively disposed on theimmune-based assay unit to allow loading of the concentrated form of theantigen from the concentrator unit to the immune-based assay unit. Insome embodiments, the antigen is TB-LAM. The immune-based assay unitcomprises one or more rapid diagnostic testing device, enzyme linkedimmune sorbent assay (ELISA), lateral flow assay, or a combinationthereof. In some embodiments, the device further comprises a dilutorunit.

Referring to FIG. 2A, a device 30, in accordance with one embodiment,comprises a concentrator unit 32 and an immune-based assay unit 34. Theconcentrator unit 32 comprises an anion exchange medium 36 toconcentrate a target biomolecule (an antigen) present in a sourcebiological sample and generate a concentrated form of the targetbiomolecule. The immune-based assay unit 34 comprises an antibody tobind the concentrated form of the target biomolecule (such as antigen)received from the concentrator unit 32. The concentrator unit 32 isoperatively coupled to the immune-based assay unit 34 to allow loadingof the concentrated form of the target biomolecule, such as antigen fromthe concentrator unit 32 to the immune-based assay unit 34. Theconcentrator unit 32 of the device 30 also includes an input channel orinlet 38 for loading the source biological sample to the ion-exchangemedium 36. The concentrator unit 32 further includes an output channelor outlet 39 for allowing the eluate to flow from the concentrator unit32 to the immune-based assay unit 34. The eluate comprises aconcentrated form of the target biomolecule. In some other embodimentsof FIG. 2A, the source biological sample may be introduced in the device30 without any dilution. In these embodiments, the source biologicalsample is contacted with the ion-exchange medium 36 without anydilution. In such embodiments of device 30, a salt-tolerantanion-exchange medium, for example, an anion exchange membranecontaining primary amine may be used.

In some embodiments, the ion-exchange medium 36 is disposed in theconcentrator unit 32 for rapid separation of the undesired constituentsfrom the biological sample and at least generates a concentrated form ofthe target biomolecules. By using the ion-exchange medium 36, aconcentration of the target biomolecule in the eluate may be increasedto 2× to 25× compared to a concentration of the target biomolecule inthe source biological sample. For example, by using a Q+ anion exchangemembrane for concentrating the target biomolecule, the TB-LAM isconcentrated by 15× and 25×, as shown in FIGS. 6 and 7, respectively, aswill be described later.

The ion-exchange medium 36 comprises one or more ligands, wherein theligands comprise cationic- or anionic-exchange ligands. Theanionic-exchange ligands include, but are not limited to, quaternaryammonium ion, and dimethyl aminoethyl (DMAE) groups.

In some embodiments, the ion-exchange medium 36 includes an ion exchangematerial, an ion-exchange membrane, or an ion-exchange matrix. Theion-exchange medium 36 may be disposed on the concentrator unit 32 forconcentrating the target biomolecule. In one embodiment, theion-exchange medium is an anion-exchange membrane, for example Q+membrane (GE Healthcare Life Sciences, Pittsburgh, Pa., US). Further,the anion exchange membrane is easily adaptable to the concentrator unitand to the immune-based assay unit, such as RDT. The anion exchangemembrane is also easy to dispose to the concentrator unit 32 in thefield-able applications. In some embodiments, an anion-exchange membranespecific for capturing TB-LAM may be used to concentrate TB-LAM beforeloading the eluate comprising the concentrated form of the TB-LAM to therapid diagnostic testing device. For example, an amine-basedion-exchange membrane is employed to concentrate TB-LAM from the urinesample. In such embodiments, the amine-based ion-exchange membrane isused for concentrating TB-LAM, which significantly improves a signal fordetection of TB.

In an alternate embodiment, the ion exchange medium 36 may comprise ananion-exchange resin. In such embodiment, a column chromatographycontaining an anion exchange resin is used to capture and concentratethe target biomolecule, such as TB-LAM. For example, Capto™ Adhere resin(GE Healthcare Life Sciences, Pittsburgh, Pa., US) or Capto™ AdhereImpRes resin (GE Healthcare Life Sciences, Pittsburgh, Pa., US) may beselected as the anion-exchange resin to capture TB-LAM. In theanion-exchange resin chromatography, a larger volume of buffer isrequired to elute the biomolecule compared to the volume of bufferrequired for anion-exchange membrane. Further, an efficient packing ofthe resin and refrigeration of the resin are required in the anionexchange resin chromatography.

Referring now to FIG. 2B, a device 40 comprises a dilutor unit 41 inaddition to the components of the concentrator unit 32, and theimmune-based assay unit 34 of FIG. 2A. The device 40 may be employed inembodiments where it is desirable to dilute the source biological samplein the device 40 before contacting the source biological sample with theion exchange medium 36. The dilutor unit 41 includes a dilutor 42 and aninput chamber 44. The input chamber 44 is configured to receive thesource biological sample. The dilutor unit 41 is coupled to theconcentrator unit 32, and operatively coupled to the immune-based assayunit 34 via the concentrator unit 32. The dilutor 42 may be a mixingchamber or mixing vessel where the dilution of the source biologicalsample takes place by mixing the source biological sample with anappropriate buffer solution. In some embodiments, the dilutor 42 may bepre-filled with a buffer solution as per requirement of the desireddilution. In some embodiments, the required volume of buffer is added tothe dilutor 42 after addition of the source biological sample to theinput chamber. The input chamber 44 is a chamber for receiving thesource biological sample and feeding the source biological sample to thedilutor 42 in a controlled manner using a plunger 45. In someembodiments, the input chamber 44 is a syringe having a plunger. Theplunger 45 may be linearly pulled and pushed along the inside of thechamber 44 in directions represented by arrows 49, allowing the chamber44 (e.g., syringe) to receive and discharge the source biological samplethrough a discharge outlet 47 located at the front end (opposite end ofthe plunger 45) of the input chamber 44 such as the syringe. Thedischarge outlet 47 may be an orifice, a membrane, or a filter throughwhich the source biological sample may pass through and enter thedilutor 42.

Referring now to both FIGS. 2A and 2B, the concentrator unit 32 receivesat least a portion of the diluted biological sample through the inlet38. The concentrator unit 32 comprises an ion-exchange medium 36 forrapid separation of undesired materials from the source biologicalsample and forms at least partially purified biological samplecomprising concentrated form of the biomolecule. The eluate comprisingthe concentrated form of the target biomolecule is directed to theimmune-based assay unit 34 through an outlet 39 of the concentrator unit32.

In some embodiments, the immune-based assay unit 34 is a lateral flowunit. The lateral flow unit comprises a sample receiving zone 46 and adetection zone 48. The sample receiving zone 46 may further be referredto as a “sample application pad” or a “sample pad.” The sample receivingzone 46 is operatively coupled to the concentrator unit 32 such that theconcentrated form of the biomolecule, such as TB-LAM is received fromthe concentrator unit 32 by the sample receiving zone 46. The samplereceiving zone 46 may be present on a fiber glass, quartz, or acellulose substrate for receiving the biological sample comprising aconcentrated form of the target biomolecule.

Optionally, a conjugate zone 49 is disposed adjacent to and upstream ofthe sample receiving zone 46, wherein the conjugate zone comprises aconjugate particle for binding with the target biomolecule. Thedetection zone 48 is disposed adjacent to and downstream of the samplereceiving zone 46. The detection zone 48 comprises at least one antibodyfor capturing the target biomolecule.

As noted, the concentrator unit 32 is operatively coupled to theimmune-based assay unit 34, such as a LFA unit, to allow loading of theconcentrated form of the antigen from the concentrator unit 32 to theimmune-based assay unit 34. The concentrator unit 32 and the lateralflow unit 34 are operatively coupled or connected at least by a fluidiccommunication. The fluidic communication facilitates a fluid flow fromthe concentrator unit 32 to the immune-based assay unit 34 such as alateral flow unit during operation of the device 30, 40. For example, aurine sample flows from the concentrator unit 32 to the lateral flowunit 34, which provides a fluidic communication under the operatingconditions of the diagnostic testing device. In some embodiments, theconcentrator unit 32 and the immune-based assay unit 34 may be inphysical contact with one another. In such embodiments, the concentratorunit 32 and the immune-based assay unit 34 may be coupled or connectedby a mechanical fastener.

In certain embodiments, the concentrator unit 32 of the device 30 or 40and the immune-based assay unit 34 are disposed at angles in a rangefrom 30 degrees to 120 degrees with respect to one another. In someembodiments, the concentrator unit 32 and the immune-based assay unit 34are vertically disposed with respect to one another. In particular, inthese embodiments, planes of the concentrator unit 32 and theimmune-based assay unit 34 are disposed at 90-degree angle with respectto one another.

As noted, immune-based assay unit, such as a rapid diagnostic testingdevice 34 comprises an antibody, wherein the antibody is specific to theantigenic moiety of the target biomolecule. For example, the rapiddiagnostic testing device comprises a TB-LAM-specific antibody. In oneor more embodiments, the antibody is a monoclonal antibody. In someother embodiments, the antibody is a polyclonal antibody. In someexamples, the polyclonal antibody is an affinity purified polyclonalantibody. Due to high structural complexity and variability of TB-LAM, acomplex spectrum of antigenic epitopes is generated. The use of affinitypurified polyclonal antibody allows to cover the full spectrum ofantigenic specificities potentially associated with LAM present inclinical samples. To achieve the desired assay sensitivity ofimmunoassay, for example in a sandwich immunoassay, the highestconcentration of antigen-specific labeled antibody may be used. Theenriched antibodies may be raised to the antigenic moiety of the TB-LAM,may be for a specific epitope of LAM in an environment which maintainsits antigenic activity.

In one or more embodiments, the TB-LAM-specific antibody used for a RDTdevice, such as immune-based assay unit 34 (e.g., LFA strip), is areporter-linked TB-LAM-specific antibody or a labeled antibody. As usedherein, “labeled antibody” includes an antibody coupled to an enzyme ora substrate. In some examples, the enzyme is capable of changing coloron exposure to a substrate. In some examples, the substrate is capableof changing color on exposure to a reagent (such as an enzyme),respectively. As such, the antibody may be labeled with a dye, a metalparticle (e.g., gold), a compound capable of producing chemiluminescenceor fluorescence. In alternative embodiments, the antibody may beattached to a magnetic bead, a cellulose bead, a polymeric bead labeledwith a dye, an affinity probe, and the like.

In some embodiments, the immune-based assay unit 34 of the device 30 or40 is a lateral flow assay (LFA) unit, an enzyme linked immuno-sorbentassay (ELISA) unit, or a combination thereof. In one embodiment, theimmune-based assay unit 34 is a LFA unit, wherein the LFAs are used fordetection of target biomolecules, such as different biomarkers presentin a source biological sample (such as urine). In some embodiments, theLFA includes dye-labeled antibodies to capture biomolecules and producea visible band on the LFA unit, such as a nitrocellulose test strip. Incertain embodiments, the target biomolecule (such as TB-LAM) binds tothe antibodies disposed on the test line forming biomolecule-antibodycomplexes, which are further bound to conjugate particle-coupledantibodies on the test line, forming a visible test line in the resultwindow. In immune-based assay unit 34, the antibodies are attached toanother line, a second line, referred to herein as a control line(referred to as “C”). This control line typically comprises aspecies-specific anti-immunoglobulin antibody, specific for theconjugate particle-coupled antibody. The control line gives informationon integrity of the conjugate particle-coupled antibody and fluidics ofthe lateral flow unit.

As illustrated in FIGS. 2A and 2B, a concentrated form of the targetbiomolecule, such as TB-LAM is received by the sample receiving zone 46.The detection zone 48 comprises at least one binding agent for detectingthe at least one biomolecule by capturing the biomolecule. The detectionzone may be constructed on a nitrocellulose membrane. In one embodiment,the detection zone may be formed by depositing one or more bindingagents on the nitrocellulose membrane. The detection zone 48 comprises atest region or test line “T.” The test region is a sub-zone of thedetection zone 48 where antibody is deposited. The detection zonefurther comprises a control region or control line “C.” One or morebinding agents having affinity towards the conjugate particles depositon the control region C, these binding agents do not exhibit anyaffinity towards the biomolecule. The target biomolecule (such asTB-LAM) binds to the antibodies disposed on the test line T formingbiomolecule-antibody complexes. The biomolecule-antibody complex mayfurther bound to conjugate particle-coupled antibodies on the test lineT, forming a visible test line in the result window (not shown in FIGS.2A and 2B). The presence of the target biomolecule is visually detectedby change in color of the test region T in the detection zone 48 on alateral flow unit. In immune-based assay unit, such as a LFA unit, theantibodies are attached to a different line other than the test line T,referred to herein as a control line C at the detection zone 48. Thecontrol line C gives information on integrity of the conjugateparticle-coupled antibody and fluidics of the lateral flow unit, as thecontrol line C typically comprises a secondary antibody specific for theconjugate particle-coupled antibody.

The conjugate particle may include colloidal gold, a colored particle, afluorescent probe, a paramagnetic particle (such as paramagneticmonodisperse latex particle), or combinations thereof. The lateral flowunit 34 may further include alternative conjugate reporters such ascellulose nanobeads (CNB), magnetic beads, fluorescence tags,chemiluminescence molecules, or various shapes of gold nanoparticlesincluding nanospheres, nanorods, and nanoshells. Such alternativeconjugate reporters are contemplated within the scope of embodimentspresented herein. The conjugate particle is conjugated to one of thecomponents of the biological sample, a component of the lateral flowassay strip (such as binding agent), or a biomolecule such as a protein.The protein may be an antigen or an antibody, depending on a format ofthe assay.

As noted above with respect to blocks 22, 24, and 26 of FIG. 1, thesource biological sample, the flow through, and the eluate,respectively, may be tested to determine presence or absence of thetarget biomolecule. Referring now to FIG. 3, three different sourcebiological samples (i), (ii) and (iii) were tested using a commerciallyavailable enzyme linked immunosorbent assay (ELISA) kit and/or acommercially available rapid diagnostic test kit. In the illustratedexample of FIG. 3, the source biological samples (i)-(iii), such asurine samples, were subjected to commercially available rapid diagnostictest kits 49, 50, and 51 before contacting the source biological samplesto respective ion-exchange media. The presence of TB-LAM in the threesource biological samples (i)-(iii) was confirmed by the presence ofbands in the rapid diagnostic test kits 49, 50 and 51. The rapiddiagnostic test kits 49, 50 and 51 showed different concentrations ofTB-LAM for the three samples (i)-(iii).

Referring now to FIG. 4, the flow through derived from the threedifferent source biological samples, such as the samples (i)-(iii) usedin the example of FIG. 3, were subjected to commercially available rapiddiagnostic test strips of the kits, such as 49′, 50′ and 51′. Absence ofany band in the rapid diagnostic test strips 49′, 50′ and 51′ for theflow through derived from the samples (i)-(iii) confirmed the absence ofTB-LAM in the flow through of each of the samples. The results shown inFIG. 4 further indicate that the substantial portion of the targetbiomolecule binds to the ion-exchange medium and therefore the targetbiomolecule is not present in the flow through.

Referring now to FIG. 5, in an example, eluates obtained from theion-exchange medium for the source biological samples (i)-(iii), weresubjected to a commercially available rapid diagnostic test strips ofthe kits 49″, 50″ and 51″. The presence of TB-LAM in each of the eluatefrom the three source biological samples (i)-(iii) was confirmed by thepresence of bands on the rapid diagnostic test strips, such as 49″, 50″and 51″ (FIG. 5). The intensity of the bands of TB-LAM on the rapiddiagnostic test kits, 49, 50 and 51 of FIG. 3 and strips 49″, 50″ and51″ of FIG. 5 was higher relative to intensity of the bands obtainedusing conventional methods. The increase in the intensity of the bandsfurther confirmed generation of higher concentration of TB-LAM in theeluate derived from the three source biological samples (i)-(iii).

FIG. 6 illustrates the results of rapid diagnostic testing of differenteluate derived from 5 different source biological samples (iv), (v),(vi), (vii) and (viii), on the rapid diagnostic test strips 52, 54, 56,58 and 60. The source biological samples (iv)-(viii) were eluted usingan ion-exchange medium followed by run on the rapid diagnostic teststrips 52, 54, 56, 58, and 60. The concentrations of TB-LAM in thesource biological samples (iv)-(viii) were 0, 0.5, 2, 4 and 6 ng/ml (notshown), respectively, wherein the concentrations of the TB-LAM in theeluate derived from the same samples (iv)-(viii) were 0, 7.5, 30, 60 and90 ng/ml on the rapid diagnostic test strips 52, 54, 56, 58, and 60,respectively. Each of the eluate derived from the samples (iv)-(viii)was concentrated to 15× by contacting the source biological samples tothe ion-exchange medium of the concentrator unit. Further, FIG. 7illustrates the results of rapid diagnostic testing of different sourcebiological samples (ix) and (x) on the test strips 62 (4 ng/ml) and 66(6 ng/ml), respectively, and their corresponding eluate loaded on thetest strips 64 (100 ng/ml) and 68 (150 ng/ml), respectively (as depictedin FIG. 7). Each of the eluate on the test strips 64 and 68 wereconcentrated to 25× by contacting the source biological samples to theion-exchange medium 36.

The methods and devices presented herein enable enhanced detection of atarget biomolecule in a source biological sample by increasing theconcentration of the target biomolecule in the biological samplecompared to the concentration of target biomolecule in a sample used forcommercially available RDT devices. In some embodiments, a volume of aurine sample generally employed for the device 30, 40 may be in a rangefrom about 2.5 ml to 10 ml. The target biomolecule present in such highvolume of the urine sample is more than that is present in a smallervolume of the urine sample. By using the concentrator unit, the targetbiomolecule is concentrated and eluted form the concentrator unit in70-100 μL volume. The ability of a device to process larger samplevolume by using a concentrator unit indicates that a larger amount oftarget biomolecule reaches the lateral flow unit while using the device,which results in improving the signal intensity of the device.Advantageously, in the present Application, the device generates ˜20×concentrated target biomolecule (or antigen), which allows enhancedintensity signals while using lower volume of the concentrated samplesthereby providing improved sensitivity of the point of care (POC) test.

In one or more embodiments, the source biological sample comprisesurine, blood, feces, sweat, saliva, mucous, milk, semen, serum, plasma,sputum, tears, tissue, or combinations thereof. For detecting surfacepolysaccharides (LAM) using different body fluids such as serum, urineor sputum have been investigated, however, none of these body fluidswere found to be effective for diagnosing extra-pulmonary mycobacterialinfections such as those on the rise in HIV-positive subjects.Embodiments of the present method overcome such difficulties byproviding enriched mycobacterial antigens TB-LAM in a wide range ofsample types from a subject. In some embodiments, the sample typesinclude unprocessed, or undiluted urine sample.

EXAMPLES

Materials: Predictor® ReadyToProcess Adsorber Q+96-well plate column(catalog #17372119) was from GE Healthcare Life Sciences, Pittsburgh,Pa., US. Predictor® ReadyToProcess Adsorber Q+96-well plate column isinterchangeably used herein as Q+ membrane column. TB-LAM antigen (fromhuman urine samples) was obtained BioReclamation IVT, Westbury, N.Y.NUNC Maxisorb 96 wells plates (Catalog no 446469-strips or456537-plates), Capture Antibody (IV-03) of 5 μg/mL; 10% BSA solution(37525), Biotinylated Detection antibody (IV-02b), SA-HRP(streptavidin—horseradish peroxidase) conjugate (Catalog no N100), and25 mL reservoirs for multichannel pippettor (Catalog no. 8093-11) werepurchased from ThermoFisher Scientific, Waltham, Mass., US. CarbonateCoating Buffer (Catalog no C3041-50CAP), PBS-T wash buffer(P3563-10PAK), 0.5 M HCl as a Stop solution (Catalog no 320331-500 ML),1M Tris, pH 8.0 (Catalog no T2694-1L) were obtained from Sigma, StLouis, Mo., US. 10×PBS (Catalog no 17-517Q) was from Lonza, Basel,Switzerland. 3,3′, 5,5′-Tetramethylbenzidine (TMB) Liquid SubstrateSystem for ELISA (Catalog no ab171523) was from abcam, Cambridge, Mass.,US. The 3,3′, 5,5′-Tetramethylbenzidine Liquid Substrate System forELISA is interchangeably used herein as a TMB substrate. Plate covers(plastic cover for short incubations) (Catalog no 353913) were fromFalcon®, Corning, N.Y., US and adhesive cover (Catalog no 60941-120) forovernight incubations was from VWR, PA, US.

Example 1: Effect of Dilution of a Source Urine Sample in ConcentratingTB-LAM Present in the Source Urine Sample Using an Ion Exchange Medium

A urine sample was spiked with TB-LAM to have a final concentration of24 ng/ml for TB-LAM in the urine sample. The urine sample was diluted to4× to reduce the conductivity to a range that is optimal for an anionexchange membrane to bind the TB-LAM antigen. 6.2 ml of the dilutedurine sample containing 6 ng/ml TB-LAM was applied to a Q+ membranecolumn, wherein the TB-LAM was captured to the Q+ membrane column (i.e.,membrane-bound TB-LAM or captured TB-LAM). The membrane-bound TB-LAM waseluted from the Q+ membrane column using 1.35 M NaCl in 20 mM Trisbuffer. 0.1 mL eluate was loaded to an RDT device, wherein the eluatecontained TB-LAM at a concentration of 315.1 ng/ml in an elution buffercontaining 1.35 M NaCl in 20 mM Tris buffer. The TB-LAM was concentratedto 51.5-fold using the Q+ membrane column. The result yields about 13×increase in concentration using a diluted urine sample (4×) over theundiluted urine sample and commensurate improvement in sensitivity ofthe RDT.

Example 2: Qualitative Analysis of Urine Sample Containing TB-LAM Beforeand after Concentrating the Urine Sample

Preparation of Q+ Membrane Columns:

Predictor® ReadyToProcess Adsorber Q+96-well plate columns (or Q+membrane columns) were prepared for binding of TB-LAM antigen from humanurine samples by washing each well with 2 mL of acidified buffersolution. After washing the Q+ membrane column, the column wasequilibrated with a neutral pH buffer solution (e.g., phosphate bufferedsaline (PBS)).

Preparation of Urine Sample Containing TB-LAM:

TB-LAM antigen was added to a urine sample to attain a TB-LAMconcentration of 24 ng/mL. The urine sample was diluted (4× dilution) togenerate a diluted urine sample having desired salinity and enableefficient binding to the anion exchange membrane (e.g., ˜10 mS/cm). Theresulting concentration of TB-LAM antigen was 6 nanogram/mL (ng/mL),which is a level in the typical range for a human TB-infected patientand in the working range of commercially available point-of-care RDTs.The urine was then filtered through a 0.2 um ePTFE filter. Theconcentration of the TB-LAM used in the following experiments were in arange of 10 picograms/mL to 240 ng/mL.

Concentration of TB-LAM of the Source Urine Sample Using Q+ MembraneColumns:

Two 10 mL aliquots and a 25-mL aliquot of diluted urine samplescontaining 6 ng/mL TB-LAM were contacted to the washed and equilibratedQ+ membrane columns for binding the TB-LAM to the Q+ membrane columns. Afraction of the flow-through from the Q+ membrane column was collectedfor determining presence or absence of TB-LAM in the flow-through. Therest of the flow through was disposed of after neutralizing the flowthrough with a bleach. In case of inefficient capture of the TB-LAM bythe column, the flow through contains TB-LAM. In case of TB-LAM bindingto the Q+ membrane column, the bound TB-LAM was eluted off from the Q+membrane column using 100 μL eluate for three times (3×100 uL). Thebound TB-LAM was eluted form the column using 1.35 M NaCl in 20 mM Trisbuffer. 84.64% of the bound TB-LAM was eluted off the membrane with thefirst 100 uL of eluate, containing 31.51 ng of TB-LAM. The remainingTB-LAM were eluted in the second (4.69 ng, 12.61%) and third (0.93 ng,2.75%) aliquots of 100 uL eluate each. The eluate containing TB-LAM wasdirectly applied to a commercially available RDT point-of-care device(or test strip), wherein the eluate contained TB-LAM in an elutionbuffer of 1.35 M NaCl in 20 mM Tris.

A commercially available RDT point-of-care test was used toqualitatively determine the presence and/or absence of TB-LAM in thesource urine sample before applied to the column (FIG. 3), flow-throughfrom the column (FIG. 4), and eluate (FIG. 5) for each sample (N=3). 60uL of each of the samples, such as source urine sample, flow-through,and eluate was applied to the commercially available RDT permanufacturer's instructions to determine TB-LAM on the test line of theRDT strip. FIG. 3 confirmed the presence of TB-LAM in the source urinesample by the generation of bands of 15 ng/ml, 42.8 ng/ml, and 60 ng/mlin the rapid diagnostic test strips 49, 50 and 51 for samples (i), (ii)and (iii), respectively. FIG. 4 showed absence of any band in the rapiddiagnostic test strips 49′, 50′ and 51′ for the flow through derivedfrom samples (i), (ii) and (iii). The results of FIG. 4 confirmed that asubstantial portion of TB-LAM of the urine sample was bound to theion-exchange medium and therefore the TB-LAM was not present in the flowthrough (FIG. 4, test strips 49′, 50′ and 51′). FIG. 5 showed thepresence of TB-LAM in each of the eluates on the rapid diagnostic teststrips, such as 49″, 50″ and 51″ from the samples (i), (ii) and (iii).

The intensity of the bands of TB-LAM on the rapid diagnostic test strips49, 50 and 51 of FIG. 3 and strips 49″, 50″ and 51″ of FIG. 5 furtherconfirmed generation of higher concentration of TB-LAM in the eluatederived from the source urine samples (i), (ii) and (iii) as compared tothe TB-LAM in the source urine samples (i), (ii) and (iii). The sourceurine sample containing 24 ng/mL TB-LAM (FIG. 3) was passed through theQ+ membrane column. A concentration of the TB-LAM in the eluatecollected from the column was determined as 315.1 ng/mL. Theconcentration of TB-LAM (315.1 ng/mL) in the eluate resulted in anoverall concentration factor of about 13×. The concentrated form ofTB-LAM ensured generating comparatively stronger response in acommercially available RDT for antigen detection (FIG. 5). The effectivevolume of the source urine sample containing TB-LAM was reduced from 6.2mL to 100 uL (eluate), which resulted in concentrating the TB-LAM in theurine sample. A sample with a low TB-LAM titer, which was previouslyundetectable in RDT would become detectable by the same RDT afterconcentrating the TB-LAM using this example. FIGS. 6 and 7 also depictedcontrol and test bands for TB-LAM (qualitative representation) in theeluates for the samples (iv)-(viii) (FIG. 6) and samples (ix) and (x)(FIG. 7).

Example 3: Quantitative Analysis of Urine Sample Containing TB-LAMBefore and after Concentrating the Urine Sample

The source urine sample before applying to the column, and the eluatefor different samples (N=2) were quantified by ELISA assay against astandard dilution series of TB-LAM in the solution used at each point ofthe assay. A quantitative estimation included determination ofconcentration of TB-LAM in the source urine sample and in eluate bystandard ELISA bio-quantification methodology as described below. Theeluate containing TB-LAM was directly applied to an ELISA reaction,wherein the eluate contained TB-LAM in an elution buffer of 1.35 M NaClin 20 mM Tris.

Preparation of Reagents for ELISA:

Carbonate Coating Buffer was prepared by using contents of 1 capsulematerial into 100 mL double distilled water (ddH₂O). Capture antibody(IV-03) was used as 5 μg/mL concentration. 10% BSA solution was dilutedto 1:10 to 1% in 1×PBS to make a Blocking Buffer. Biotinylated antibodywas used at a concentration of 0.3 μg/mL in 1×PBS. 1 mg/mL TB-LAM wasused for various examples. 10×PBS was diluted in ddH₂O in a dilution of1:10 to form 1×PBS for use as a diluent buffer. For preparing 1×PBS-Twash buffer, content of PBS-T buffer package (as purchased) wasdissolved in 1 L ddH₂O. The TMB substrate was used as received frommanufacturer. 0.5 M HCl was used as a Stop solution. A concentrated(37%) HCl was added to ddH₂O in a ratio of 1:24 to prepare a 0.5M HCl.1M Tris, pH 8.0 was used to normalize pH of urine samples to pH 8.

ELISA Detection Method:

Absorbance-based microplate reader, which can read absorbance at 450 nmand preferably at 550 nm was used for ELISA detection. Distilled ordeionized water (ddH₂O) was used for dilution of the samples or cleaningof tubes or plates. 96-well plate washer (e.g. TECAN) was used formanually washing the plates, if necessary. 12- or 8-channel 200 μLpipettor, 25 mL reservoirs for multichannel pippettor were used for theassays.

Wash Plate Procedure:

Liquid from each well of the 96-well plate was aspirated and discardedeither manually or with an automated plate washer followed by dispensing300 μL of PBS-T into each well. Dispensing and/or aspiration wererepeated for at least two times such that wells had 3×300 μL PBS-Twashes. After final aspiration, the assay constituents were added to thewells promptly to prevent drying out the wells.

General Protocol for ELISA:

Test samples used for ELISA assay were the TB-LAM spiked urine sample,eluate from the Q+ column for the respective source urine samples. Thecontrol sample was an elution buffer (1.35 M NaCl in 20 mM Tris). 100 μLof capture antibody (IV-02) diluted in a carbonate buffer was added tothe bottom of each well of a 96-well plate. The 96-well plate was sealedbefore incubation at 4° C. for overnight. After incubation, 200 μL ofblocking buffer was added to each well of the 96-well plate for blockingthe antibodies at the bottom of the wells. The 96-well plate was coveredand incubated at room temperature for 1 hour. 100 μL of prepared testand control samples were added to the wells of the 96-well platecontaining the capture antibodies. The 96-well plate was covered andincubated at room temperature for 1 hour for binding the TB-Lam of thetest sample to the capture antibody. 100 μL of diluted detectionantibody (IV-02b @ 0.3 μg/mL in 1×PBS) was added to each of the wellsfor detecting the TB-LAM-bound capture antibody. The plate was coveredand incubated at room temperature for 45 minutes for complete binding ofthe detection antibody to the TB-LAM-bound capture antibody. 100 μL ofdiluted SA-HRP (Streptavidin-HRP) conjugate was added to each well,wherein the SA-HRP was diluted in a ratio of 1:7,500 in 1×PBS. TheSA-HRP conjugate was used for binding to biotinylated detectionantibody. The 96-well plate was covered and incubated at roomtemperature for 30 minutes. A stop solution and a TMB ELISA substratesolution were prepared as per manufacturer's direction. 100 μL of TMBELISA substrate was added to each well of the 96-well plate withoutcross contaminating the reservoir. The TMB ELISA substrate generallydetects horseradish peroxidase (HRP) activity. Unreacted substrate wastypically colorless or generated very light yellow color. When the TMBELISA substrate system was reacted with peroxidase, a blue-coloredsoluble reaction product was developed in the wells. The reaction wasstopped using a stop solution, producing a soluble yellow reactionproduct, which was stable for at least 1 hour.

A 96-well plate on a white background was developed on the bench andincubated for about 4-6 minutes, such that a blue color should beclearly visible. Stop solution was added if any well for blank reactionstarts forming blue color. 100 μL of stop solution was added to eachwell in the same order as added to the TMB ELISA substrate, wherein thewells with blue color changed to bright yellow. The plate was evaluatedwithin 30 minutes of stopping the reaction. The absorbance of each wellwas measured at 450 nm and 550 nm. The absorbance value at 550 nm wassubtracted from the absorbance value at 450 nm to correct opticalimperfections in the microplate. A curve-fitting statistical softwarewas used to plot a four-parameter logistic curve fit to the standardsand then calculated results for the test samples.

Determination of Concentration of TB-LAM in Urine Sample:

A concentration of TB-LAM in an eluate collected from a Q+ membranecolumn was determined quantitatively using an ELISA kit. Theconcentrations of TB-LAM in the source biological samples (iv), (v),(vi), (vii) and (viii) (referred to herein as (iv)-(viii)) weredetermined by ELISA as 0, 0.5, 2, 4 and 6 ng/ml (data not shown). Theconcentrations of TB-LAM in the corresponding eluates were determined byELISA as 0, 7.5, 30, 60 and 90 ng/ml. The same eluates of samples(iv)-(viii) were run on the rapid diagnostic test strips 52, 54, 56, 58,and 60, respectively (FIG. 6). TB-LAM in the eluates derived from thesamples (iv)-(viii) were concentrated to 15× by contacting the sourceurine samples to the Q+ membrane column of the concentrator unit.

Further, the concentrations of TB-LAM were determined as 4 and 6 ng/mlin source urine samples (ix) and (x) respectively and 100 and 150 ng/mlin the eluates corresponding to the same samples (ix) and (x),respectively. The source urine samples (ix) and (x) and correspondingeluates were also applied to the rapid diagnostic testing strips 62, 64,66, and 68, as shown in FIG. 7. Each of the eluate on the test strips 64(100 ng/ml) and 68 (150 ng/ml) were concentrated to 25× by contactingthe source biological samples 62 (4 ng/ml) and 66 (6 ng/ml) to theion-exchange medium.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedembodiments are intended to cover all such modifications and changes asfall within the scope of the invention.

1. A rapid detection method of a target biomolecule, comprising:providing a source biological sample comprising the target biomolecule,wherein the target biomolecule comprises an antigenic moiety; contactingthe source biological sample to an ion-exchange medium comprising one ormore ligands to capture the target biomolecule and form acaptured-biomolecule; eluting the captured-target biomolecule from theion-exchange medium as an eluate comprising a concentrated form of thetarget biomolecule in a solution, wherein a concentration of the targetbiomolecule in the eluate is in a range from about 2× to 25× compared toa concentration of the target biomolecule in the source biologicalsample, and wherein the solution has a salt concentration greater than150 mM; and loading the eluate comprising the concentrated form of thetarget biomolecule in the solution to a rapid diagnostic testing devicecomprising an antibody, wherein the target biomolecule binds to theantibody under the salt concentration of greater than 150 mM.
 2. Themethod of claim 1, wherein the target biomolecule is a glycolipid. 3.The method of claim 1, wherein the target biomolecule is atuberculosis-lipoarabinomannan (TB-LAM).
 4. The method of claim 1,wherein the target biomolecule binds to the antibody under the saltconcentration in a range from about 0.5M to about 2M.
 5. The method ofclaim 1, wherein the target biomolecule binds to the antibody under thesalt concentration of about 1.3M.
 6. The method of claim 1, wherein theelution of the captured-target biomolecule from the ion-exchange mediumis effected under a salt concentration in a range from about 0.5M toabout 2M.
 7. The method of claim 1, wherein the eluate comprising theconcentrated form of the target biomolecule is loaded without anydilution to the rapid diagnostic testing device comprising an antibody.8. The method of claim 1, further comprising diluting the sourcebiological sample comprising the target biomolecule by at least 2× toform a diluted biological sample.
 9. The method of claim 1, furthercomprising diluting the source biological sample comprising the targetbiomolecule by 4× to form a diluted biological sample.
 10. The method ofclaim 1, wherein the rapid diagnostic testing device comprises a lateralflow assay (LFA) device, an enzyme linked immuno-sorbent assay (ELISA)device, or a combination thereof.
 11. The method of claim 10, whereinthe rapid diagnostic testing device comprises a lateral flow assay (LFA)device.
 12. The method of claim 1, wherein the source biological samplecomprises urine, blood, feces, sweat, saliva, mucous, milk, semen,serum, plasma, sputum, tears, tissue, or combinations thereof.
 13. Themethod of claim 12, wherein the source biological sample is urine. 14.The method of claim 1, wherein the ion-exchange medium comprises an ionexchange material, an ion-exchange membrane, or an ion-exchange matrix.15. The method of claim 1, wherein the one or more ligands compriseanionic ligands.
 16. The method of claim 15, wherein the anionic ligandsare quaternary ammonium ion, or dimethyl aminoethyl (DMAE) groups.
 17. Amethod for rapid diagnostic testing of a source urine sample comprisingtuberculosis-lipoarabinomannan (TB-LAM), comprising: (a) concentratingthe TB-LAM by: diluting the source urine sample by at least 2× comparedto the source urine sample to form a diluted urine sample; contactingthe diluted urine sample to an anion-exchange medium to capture theTB-LAM of the diluted urine sample; capturing the TB-LAM of the dilutedurine sample by the anion-exchange medium; and eluting thecaptured-TB-LAM from the anion-exchange medium as a concentrated form ofTB-LAM in an eluate under a salt concentration of at least 1M, wherein aconcentration of the TB-LAM in the eluate is in a range from about 2× to25× compared to a concentration of the TB-LAM in the diluted urinesample; and (b) loading the eluate comprising the concentrated form ofthe TB-LAM of step (a) to a lateral flow assay device comprising aTB-LAM-specific antibody for binding the concentrated form of theTB-LAM, wherein the eluate is loaded without any dilution, and whereinthe TB-LAM binds to the TB-LAM-specific antibody under the saltconcentration of at least 1M.
 18. The method of claim 17, wherein theTB-LAM-specific antibody is a reporter-linked TB-LAM-specific antibody.19. The method of claim 18, further comprising generating a signal onbinding of the TB-LAM to the TB-LAM-specific antibody in case ofexceeding a pre-determined threshold value for a concentration of theTB-LAM.
 20. A device, comprising: a) a concentrator unit comprising ananion exchange medium to concentrate an antigen from a biological sampleand generate a concentrated form of the antigen; and b) an immune-basedassay unit comprising an antibody to bind the concentrated form of theantigen received from the concentrator unit, wherein the concentratorunit is operatively coupled to the immune-based assay unit to allowloading of the concentrated form of the antigen from the concentratorunit to the immune-based assay unit.
 21. The device of claim 20, whereinthe antigen is TB-LAM.
 22. The device of claim 21, wherein theimmune-based assay unit comprises rapid diagnostic testing device,enzyme linked immune sorbent assay (ELISA), or lateral flow assay. 23.The device of claim 20 further comprising a dilutor unit.